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Published on behalf of the European Society of Cardiology. All rights reserved.
© The Author 2013. For permissions please email: [email protected]
NO-dependent CaMKII activation during β-adrenergic stimulation
of cardiac muscle
D. Gutierrez, M. Fernandez-Tenorio, J. Ogrodnik, E. Niggli
Department of Physiology, University of Bern, Bern, Switzerland
Corresponding author:
Ernst Niggli, MD
Department of Physiology
University of Bern
Bühlplatz 5
CH-3012 Bern
Switzerland
Fax: +41 31 631 4611
Tel: +41 31 631 8730
Email: [email protected]
Cardiovascular Research Advance Access published August 20, 2013 at Fachbereichsbibliothek on A
ugust 20, 2013http://cardiovascres.oxfordjournals.org/
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source: https://doi.org/10.7892/boris.45834 | downloaded: 7.12.2020
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Abstract
Aims: During β-adrenergic receptor (β-AR) stimulation, phosphorylation of
cardiomyocyte ryanodine receptors (RyRs) by protein kinases may contribute to an
increased diastolic Ca2+ spark frequency. Regardless of prompt activation of PKA during
β-AR stimulation, this appears to rely more on activation of CaMKII, by a not yet
identified signaling pathway. The goal of the present study was to identify and
characterize the mechanisms which lead to CaMKII activation and elevated Ca2+ spark
frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions.
Methods and results: Confocal imaging revealed that β-AR stimulation increases
endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of
CaMKII and a subsequent increase of diastolic Ca2+ spark frequency. These changes of
spark frequency could be mimicked by exposure to the NO donor GSNO and were
sensitive to the CaMKII inhibitors KN-93 and AIP. In-vitro, CaMKII became nitrosated
and its activity remained increased independent of Ca2+ in the presence of GSNO, as
assessed with biochemical assays.
Conclusions: β-AR stimulation of cardiomyocytes may activate CaMKII by a novel
direct pathway involving NO, without requiring Ca2+ transients. This crosstalk between
two established signaling pathways may contribute to arrhythmogenic diastolic Ca2+
release and Ca2+ waves during adrenergic stress, particularly in combination with cardiac
diseases. In addition, NO dependent activation of CaMKII is likely to have repercussions
in many cellular signaling systems and cell types.
Keywords: CaMKII; Ca sparks; Ca waves; NO-synthase
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1. Introduction
In cardiac excitation-contraction coupling (EC-coupling) Ca2+-induced Ca2+ release
(CICR) is the mechanism that amplifies the Ca2+ signal initiated by entry of Ca2+ via
voltage-dependent Ca2+ channels.1 During each systole, CICR generates a robust Ca2+
transient by releasing Ca2+ from the sarcoplasmic reticulum (SR) via Ca2+ release
channels (a.k.a. ryanodine receptors or RyRs). These are macromolecular complexes
located in diadic clefts, microdomains of junctional SR, in close apposition to L-type Ca2+
channels.2 Each group of RyRs and L-type Ca2+ channels forms a unit called couplon
which can create elementary Ca2+ release events, Ca2+ sparks.3 Faithful RyR functioning
is crucial in the context of cardiac muscle Ca2+ release, its synchronization and strength
of contraction. Disturbances of these elementary mechanisms have been observed
during various cardiac pathologies.4,5
Upon β-adrenergic receptor (β-AR) stimulation during emotional stress or physical
exercise, key proteins of Ca2+ signaling, such as the L-type Ca2+ channels, the RyRs and
phospholamban are phosphorylated by protein kinases,6,7 enhancing Ca2+ cycling. There
is recent evidence indicating that elevated phosphorylation levels of the RyR mediated
by protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)
may increase their activity8-11 (for reviews see7,12). During chronic β-AR stimulation, this
could constitute a Ca2+ leak and deplete the SR of Ca2+, which would reduce the
amplitude of Ca2+ transients, eventually contributing to weak heartbeats.13
Experimentally, it has proven difficult to clearly assign a role for the different kinases in
modulating RyR behavior. The generation of various transgenic mouse lines, specifically
targeting phosphorylation by PKA and CaMKII, has not clarified the situation and
provided apparently contradictory results and interpretations. However, analysis of Ca2+
spark frequencies in transgenic mice expressing constitutively phosphorylated RyRs
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(S2808D, S2814D) suggested that PKA and CaMKII-dependent phosporylation may
increase resting Ca2+ spark frequencies.5,9
Here we examined Ca2+ sparks as signals closely reflecting RyR open probability in their
native environment. While there is controversial literature on the relative importance of
PKA and/or CaMKII-dependent RyR phosphorylation,8,14 only few studies specifically
investigated changes of Ca2+ spark frequencies resulting from β-AR stimulation during
diastole or in resting cardiomyocytes.5,9,15,16 Based on these findings, it has been
suggested that PKA-dependent phosphorylation of RyRs may not be significantly
involved in the observed increase of the resting Ca2+ spark frequency. It has been
reported that this entirely hinged on the SERCA stimulation resulting from
phosphorylation of phospholamban17 or occurred via a pathway that could involve
CaMKII.14,16,18-20 Since the CaMKII-dependent increase of spontaneous spark
frequencies was observed in resting cardiomyocytes without detectable Ca2+ signals,
which are typically required for significant Ca2+-dependent activation of CaMKII,21,22 it
remained unresolved by which pathway(s) CaMKII would become activated under these
conditions.
The current study represents an effort to reveal the mechanism involved in the activation
of CaMKII in resting cardiomyocytes during β-AR stimulation, and to identify an
alternative pathway that could underlie the observed increase in Ca2+ spark frequency.
In the literature, several possible mechanisms have been mentioned. The “exchange
protein activated by cAMP” (Epac) may participate in the modulation of RyR open
probability, either directly or via CaMKII.18 Another alternative is activation of CaMKII by
reactive oxygen species (ROS), which is known to occur independently of detectable
Ca2+ signals.23 Therefore, we carried out experiments investigating Ca2+ sparks and
CaMKII activity to characterize the putative involvement of these and other cellular
signaling pathways.
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Together our findings reveal that upon β-AR stimulation CaMKII becomes activated in a
manner that does not require Ca2+ transients, initiated by formation of endogenous nitric
oxide (NO). This represents an unexpected and newly discovered mode of CaMKII
activation occurring in parallel to PKA. Preliminary findings have previously been
presented in abstract form.24
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2. Methods For additional information on methods, see Supplementary information online.
2.1 Isolation of Guinea-pig ventricular myocytes
For all electrophysiological and confocal Ca2+ and NO imaging experiments, we used
freshly isolated Guinea-pig ventricular cardiomyocytes16 following the animal handling
procedures conforming with the National Institutes of Health Guide for the Care and Use
of Laboratory Animals (1996) and with the permission of the State Veterinary
Administration and according to Swiss Federal Animal protection law (permit BE97/09).
Animals were euthanized by stunning and cervical dislocation, followed by rapid removal
and enzymatic dissociation of the cardiac tissue.
2.2 Experimental solutions
All drugs, inhibitors and donors used during our experiments were freshly prepared daily
from aliquots. Extracellular and intracellular (patch pipette) solutions were used from a
ready made stock.16
2.3 Electrophysiological recordings
ICa recordings were carried out in the whole-cell configuration of the patch clamp
technique, resting membrane potential was set at -80mV during Ca2+ imaging.16
2.4 SR Ca2+ content preconditioning
To ensure a constant SR Ca2+ content, we performed a SR Ca2+ loading protocol with a
train of 20 membrane depolarizations from -80 to 0 mV (see Figure 1A).
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Pharmacological interventions were applied, as indicated. Changes in SR Ca2+ content
were compared to control conditions without the drug, after an identical loading protocol.
In all cases content was estimated by recording a Ca2+ transient triggered with caffeine.
2.5 Confocal Ca2+ and NO imaging
The Ca2+ spark frequency and SR Ca2+ content are shown after normalization and
expressed as mean values ±S.E.M. For these recordings we used fluo-3 as Ca2+
indicator. NO measurements were carried out using DAF-2DA. Confocal imaging was
performed with either a FluoView-1000 (Olympus) or a MRC-1000 confocal laser-
scanning microscope (Bio-Rad). Indicators were excited at 488 nm with a solid-state
laser (Sapphire 488-10) and fluorescence was detected > 515 nm.
2.6 In-vitro CaMKIIδ activity and nitrosation assays
Ca2+-independent, H2O2 and NO-dependent CaMKII activities were assessed by using
an ELISA kit. Values are shown normalized to the maximal CaMKII activation levels
reached in low Ca2+ (<10 nM Ca2+; CaM/EGTA). Nitrosation of CaMKII was quantified
using an antibody specifically detecting S-nitrosated cysteines.
2.7 Statistics
Paired or unpaired Students t-tests were applied as appropriate to determine
significance. In figures p values of < 0.05 or < 0.01 are indicated by * or **, respectively.
N refers to number of animals, n to number of cells.
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3. Results
3.1 Ca2+ spark frequency during β-AR stimulation is modulated by CaMKII but not
PKA
We investigated cAMP-dependent pathways (e.g. PKA, Epac) to determine the
involvement of PKA and/or CaMKII in the modulation of resting Ca2+ spark frequencies.
Constant SR loading was achieved with Ca2+ preloading involving a train of L-type Ca2+
currents. SR Ca2+ content was estimated with caffeine before and after the experiment in
each cardiomyocyte (see Figure 1A and16). As shown in Figure 1B and 1C, superfusion
of resting cells with 1 µM isoproterenol (Iso) increased the frequency of Ca2+ sparks
around 4-fold within 3 min, without significantly altering the [Ca2+]SR in this time window
(see Figure 1E and16). Next, cAMP was raised independently of the β-AR receptors by
direct activation of adenylate cyclase with forskolin.11 Surprisingly, and unlike Iso, 1 µM
forskolin did not change the Ca2+ spark frequency significantly, even though the SR
content increased to 125 %, presumably resulting from SERCA stimulation without
activation of sparks in parallel. Importantly, both drugs resulted in almost identical
amplification of the L-type Ca2+ current (ICa), confirming that forskolin activated PKA to
an extent corresponding to Iso application (Figure 1D). Inclusion of the specific PKA
inhibitory peptide PKI in the patch solution did not prevent the increase in the Ca2+ spark
frequency, but resulted in a drop of SR Ca2+ content, presumably resulting from the
suppression of SERCA stimulation during Iso.
These results indicate that acute β-AR stimulation increases Ca2+ spark frequency,
independently of cAMP and therefore makes an involvement of PKA and Epac very
unlikely. Hence, we did not follow up on either of these pathways. A potential alternative
pathway could be CaMKII, despite the fact that in quiescent cells, there were no Ca2+
signals that could activate this kinase. To confirm involvement of CaMKII, as also
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suggested by our previous study,16 we carried out experiments in the presence of either
KN-93 or KN-92 or included 10 µM of the specific CaMKII inhibitor autocamtide-2-related
inhibitory peptide (AIP) in the patch solution. KN-93 and AIP prevented the increase in
Ca2+ spark frequency in Iso without changing the SR Ca2+ content (Figure 2A-C). KN-92,
the inactive negative control for KN-93, did not suppress the increase in spark frequency,
as expected. Thus, under these experimental conditions, we can use the Ca2+ spark
frequency as biological indicator for CaMKII activity.
3.2 ROS scavengers fail to prevent the increase in Ca2+ spark frequency
We then examined whether Ca2+-independent CaMKII activation by ROS23 could
underlie the higher spark frequency. For this we used Mn-TBAP, a superoxide dismutase
(SOD) mimetic, which has previously been shown in our experiments to reliably
suppress Ca2+ sparks initiated by oxidative stress.25 Interestingly, 100 µM Mn-TBAP
failed to prevent the increase in Ca2+ spark frequency and did not significantly alter SR
Ca2+ load (Figure 2D-F), indicating that CaMKII activation by β-AR stimulation is not
ROS-dependent.
3.3 NO modulates Ca2+ spark frequency upon β-AR stimulation
It has been suggested that after β-AR stimulation the increase in SR Ca2+ leak,
determined with a dedicated leak protocol, is dependent on NO production and
independent of PKA, because NO-synthase (NOS) inhibition prevented the leak
observed in the presence of Iso.11,26 To examine whether a similar mechanism could be
involved in the higher frequency of Ca2+ spark observed here, we inhibited the synthesis
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of NO while stimulating the cardiomyocytes with Iso, analogous to experiments
described in Figure 1. Pretreatment with 500 µM of the NOS inhibitor L-NAME prevented
a significant increase in Ca2+ spark frequency (Figure 3A), indicating that upon β-AR
stimulation, the spark frequency is modulated by endogenous NO produced by NOS of
the cardiomyocyte.
To confirm NO involvement, cells were loaded with diaminofluorescein (DAF-2) by
exposure to the ester (DAF-2DA; 0.1 µM). After application of Iso, we recorded a
substantial increase in DAF-2 fluorescence (19% ± 5%; Figure 3C and E). In some cells
the NO donor S-Nitroso-L-glutathione (GSNO; 500 µM) was added at the end of the
protocol, to confirm that DAF-2 resolves NO signals (Figure 3D). Please note that these
recordings were corrected for dye bleaching and should not be taken quantitatively for
NO concentrations. Because DAF-2 has been reported to detect other types of reactive
species27, we repeated the same experiment in 500 µM L-NAME to suppress NO
formation. This prevented the increase in DAF-2 fluorescence (Figure 3E and 3F, orange
symbols and columns), confirming that the signal reflects NO production. Cellular ROS
production induced by GSNO was excluded with the ROS sensitive fluorescent indicator
CM-H2DCF (see supplementary Figure S1). Together these findings firmly establish a
link between Iso-induced production of intracellular NO by the cardiomyocytes and the
observed increase of Ca2+ spark frequencies mediated by CaMKII. This interpretation is
in line with the inability of Mn-TBAP to prevent the increase in Ca2+ spark frequency
(Figure 2D and 2E) and is consistent with the observation that Mn-TBAP does not
significantly scavenge NO.28
3.4 The NO donor GSNO reproduces the increase in Ca2+ spark frequency induced
by β-adrenergic stimulation
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NO can affect the function of proteins, including the RyRs, via several known
pathways.29-31 This can occur directly as post-translational protein modifications, such as
S-nitrosation (also referred to as S-nitrosylation or transnitrosylation).12,32 Alternatively,
NO can lead to the formation of cGMP and activation of protein kinase G (PKG), which
has been shown to phosphorylate RyRs, but so far only in-vitro.33 The findings in Figure
3 indicate that after Iso application, NO mediates the increase in Ca2+ spark frequency.
To support this interpretation, we used a NO donor instead of Iso. 150 µM GSNO
resulted in a 3.22 (± 0.31)-fold increase in Ca2+ spark frequency, thereby quantitatively
mimicking the changes observed in Iso (Figure 4A and 4B). Note that in these
experiments SR Ca2+ content did not maintain the control level, presumably because
GSNO increased the Ca2+ spark frequency without concomitant SERCA stimulation
(Figure 4C). To distinguish between a direct S-nitrosation of the RyRs and an indirect
modification via CaMKII (suggested by the findings above), we tested whether AIP could
prevent the GSNO-dependent occurrence of sparks, similar to what was observed in Iso.
In the presence of AIP, GSNO did not significantly elevate spark frequency (Figure 4B).
These findings confirm that the higher spark frequency in GSNO resulted largely from
activation of CaMKII and not from a direct S-nitrosation of the RyRs or activation of PKG.
In summary, these data indicate that NO can activate CaMKII, in the absence of Ca2+
signals.
3.5 Quantification of NO-dependent CaMKII activation in-vitro
To confirm that NO could activate CaMKII in the absence of elevated Ca2+, we used an
in-vitro assay.34 CaMKII activity was detected with an ELISA test and normalized to that
observed in low Ca2+ (< 10 nM; Ca2+, CaM and EGTA, Figure 4D). As already
established,23 addition of H2O2 activated CaMKII in low Ca2+. The activity observed in 1
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µM H2O2 was 2.65 (± 0.47)-fold higher than in control. The NO donor GSNO resulted in
comparable CaMKII stimulation of 2.31 (± 0.39)-fold. CaMKII activity under these
oxidative and nitrosative conditions represented ~16% of the maximal Ca2+/CaM
dependent activity (250 µM Ca2 and 120 nM calmodulin). This confirms a direct
activation of CaMKII by NO, as suggested by our findings in cardiomyocytes.
Each CaMKII monomer is predicted by GPS-SNO software35 to have 3 potential sites for
S-nitrosation (Figure 4E upper panel). We used an antibody specifically recognizing S-
nitrosated cysteines to quantify CaMKII nitrosation after pre-incubation of CaMKIIδ with
GSNO (Figure 4E lower panel). Indeed, a 32.1% ± 13% increase of CaMKII nitrosation
was observed with this assay.
3.6 GSNO leads to arrhythmogenic Ca2+ signals In beating cardiomyocytes
At the cellular level, spontaneous Ca2+ waves (SCWS) are considered to be indicators
for arrhythmogenic conditions. Since the elevated Ca2+ spark frequencies shown above
could result in SCWS, we tested the arrhythmogenic potential of the NO donor in field
stimulated cardiomyocytes (Figure 5). In these experiments, NO presumably modifed
several relevant Ca2+ signaling proteins and membrane channels. However, the
recordings revealed an increase in the diastolic Ca2+ spark frequency, similar to what
was observed in resting cells. Furthermore, after a train of 10 depolarizations, 13.3% of
the control myocytes exhibited SCWS, while in the presence of GSNO 77.8% showed
waves. This elevated wave frequency was accompanied by a reduced SR content (to
77% ± 6.5% of control), confirming that the waves were resulting from altered function of
the RyRs and not from SR Ca2+ overload.
Taken together, our results provide compelling evidence that the observed increase in
Ca2+ spark frequency upon β-AR stimulation results from an activation of CaMKII, which
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is mediated by NO but is not dependent on Ca2+ transients (see Figure 6 for a diagram of
this pathway). This represents a new mechanism for CaMKII activation that may have far
reaching implications.
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4. Discussion
RyRs have attracted considerable research interest, due to discoveries such as RyR
mutations causing life threatening arrhythmias.36 alterations of their behavior during
several diseases, which are often caused by posttranslational modifications, most
notable phosphorylation and oxidation/nitrosation (for review see12). The participation of
the RyRs in diseases such as catecholaminergic polymorphic ventricular tachycardias
(CPVTs) and heart failure suggests that they may be potential drug targets.37,38
4.1 Modulation of RyR function
Changes of RyR function are expected to have a significant impact on cardiac Ca2+
signaling. Several laboratories have examined functional consequences of RyR
phosphorylation on various levels of complexity, from single channels to isolated cells,
partly using transgenic animal and disease models.7,39,40 These studies have resulted in
a considerable controversy and confusion regarding the functional role of the involved
protein kinases PKA and CaMKII.14,41 The reasons for this are not clear, but may arise
from different disease models, protocols and experimental designs. As suggested by the
present study, they may partly arise from unexpected cross-talks between complex
cellular signaling pathways.
4.2 Modes of CaMKII activation
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When examining the importance of protein kinases for changes of diastolic Ca2+ spark
frequencies after β-AR stimulation, we made a surprising observation. Even though there
were no visible Ca2+ signals in resting cells that could lead to significant CaMKII
activation, the increase in Ca2+ spark frequencies could be prevented by
pharmacologically blocking CaMKII (but not PKA). This immediately raised the question
how under these circumstances CaMKII could become activated? In the literature,
several possibilities have been reported, including a pathway involving “exchange factor
directly activated by cAMP” (Epac),15,42 or requiring oxidative modification of the
CaMKII.23 The observation that the application of forskolin did not elevate the propensity
of diastolic Ca2+ sparks, unlike β-AR stimulation, is in line with our conclusion, based on
the experiments with protein kinase inhibitors for CaMKII and PKA, that any PKA
involvement is highly unlikely. Furthermore, the negative result with forskolin regarding
spark frequencies makes activation of CaMKII via the Epac pathway improbable and is
consistent with the finding that forskolin does not increase SR Ca2+ leak.11 Therefore, we
carried out experiments to test for the second possibility of Ca2+ spark activation,
oxidative stress. Of note, redox modifications of the RyRs are well known to increase
their openings43,44 although direct RyR oxidation would not be sensitive to the CaMKII
inhibitor AIP, as observed in this study. Thus, we were left with the alternative that an
oxidative modification of CaMKII could be responsible for its activation. However, we
were unable to prevent the Ca2+ sparks with Mn-TBAP, a SOD mimetic which we found
to reliably suppress ROS induced sparks in a model of oxidative stress.25 This finding
suggests that ROS generation after β-AR stimulation is not involved in CaMKII activation
and is consistent with a recent report showing that ROS production does not increase
during β-AR stimulation of resting cells.45
4.3 NO activates CaMKII in cardiomyocytes
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An interesting observation has been reported in a study examining SR Ca2+ leaks in
cardiomyocytes during β-AR stimulation using a dedicated leak protocol.11 In this study,
the SR Ca2+ leak has been quantified from the drop of the cytosolic Ca2+ concentration
after blocking the RyRs with tetracaine. These authors showed that the function of
CaMKII, but also NO synthases, were important determinants for the SR Ca2+ leak.26
Surprisingly, our experiments along these lines revealed that inhibition of NOS also
prevented the increase in spark frequency, suggesting an involvement of NO signaling.
To confirm a key role of NO we used a multi-pronged approach. We were able to detect
endogenous NO production by the cardiomyocytes upon Iso application, while the NO
donor GSNO mimicked the effects of Iso on the spark frequency. In contrast, the GSNO
effect was almost completely inhibited by the specific CaMKII inhibitor AIP, suggesting
that direct RyR nitrosation was not involved. Rather this appeared to be mainly mediated
by CaMKII. Could it be that NO maintains CaMKII active at resting Ca2+ concentrations,
similar to what has been reported for ROS?23 For this to occur, an initial Ca2+-dependent
activation of CaMKII is required, which could be mediated by invisible Ca2+ signals, such
as the Ca2+ quarks suggested to underlie a fraction of the SR Ca2+ leak.46,47 To address
the intriguing question of NO-dependent CaMKII activation directly, we applied a
biochemical in-vitro assay of CaMKII activity. The results obtained confirmed that NO
can maintain CaMKII active to an extent similar to H2O2, without requiring elevated Ca2+
concentrations. Since this occurred in-vitro and was initiated by significant S-nitrosation
of the CaMKII protein, it seems very likely that NO can directly activate CaMKII.
Interestingly, a strikingly different regulation by NO and ROS has been reported for the
CaMKIIα isoform of this kinase in pituitary tumor GH3 cells. This isoform was inhibited
after nitrosation, but became activated in reducing conditions.48
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4.4 Relevance of NO-dependent CaMKII activation
Taken together, these findings provide compelling evidence for a stimulation of
endogenous NO generation by cardiomyocytes upon β-AR stimulation. In the beating
heart, this presumably additive mechanism may be even more pronounced, since further
CaMKII activation will occur by enhanced Ca2+ transients. Several studies have
previously reported positive or negative inotropic effects of NO, which may be partly
related to modifications of CaMKII and RyR function, involved NOS isoforms, or
crosstalk between activated pathways.49,50 Related to this, it has been suggested that
NO may have a biphasic effect on RyR open probability, depending on the extent of pre-
existing β-AR stimulation.51 Although the precise mechanism of this phenomenon
remains unresolved, it may involve CaMKII and could be related to changes of the
nitroso/redox balance, as several posttranslational modifications will modulate RyR
function in an exceedingly complex fashion.52
The pathway characterized here represents a newly discovered mechanism for CaMKII
activation. This results in a surge of diastolic Ca2+ sparks and increased wave
propensity. Since Ca2+ sparks are signals faithfully reporting the function of the RyRs,
this likely occurs via CaMKII-dependent modulation of the RyRs. In addition, the direct
activation of CaMKII by NO observed here and the pathways participating downstream
of CaMKII are expected to have additional repercussions for cardiomyocyte Ca2+
signaling. For example, NO mediated CaMKII activation contributes to the modulation of
Ca2+ cycling upon β-AR stimulation, such as during physical exercise or emotional
stress. During sustained β-AR stimulation, such as during heart failure, this could lead to
SR Ca2+ depletion, weaker heart beat and arrhythmias.40,53 The additional CaMKII
activation prompted by NO may be particularly detrimental if it occurs in conditions with
already hypersensitive RyRs, for example in the presence of CPVT mutations or
oxidative RyR modifications.
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Finally, the identification of this pathway adds to the experimental complexity of studies
with cardiac muscle because it represents a possibility for crosstalk between PKA and
CaMKII activation, downstream of β-AR stimulation. The existence of such a cross-talk
may explain some of the controversial experimental results and interpretations regarding
the regulation of the RyRs by PKA or CaMKII respectively.
Funding
This work is supported by grants from the Swiss National Science Foundation (31-
132689 and 31-109693 to E.N.) and by an Instrumentation Grant of the Medical Faculty,
University of Bern.
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Acknowledgements
We would like to thank Dr. N. Ullrich and I. Marcu for experimental support, as well as D.
Lüthi and M. Känzig for technical assistance.
Conflicts of interest
None.
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Figure Legends
Figure 1 The increase of Ca2+ spark frequency by Iso is mediated by β-adrenergic
receptors but not by cAMP. (A) The experimental protocol used trains of depolarizations
to load the SR with Ca2+ and caffeine to estimate SR Ca2+ content (for details see
Supplementary online information). (B) Confocal line-scan images showing Ca2+ sparks
in control solution (Ctrl) and after ~3 min of 1 µM Iso or 1 µM forskolin. (C), Normalized
Ca2+ spark frequency in Ctrl, after ~3 min of Iso (n=8, N=8) or forskolin (n=6, N=2) and
after Iso in the presence of the PKA inhibitor PKI (n=7, N=3), respectively. In control,
Ca2+ sparks are relatively indistinct and sparse in resting guinea pig cardiomyocytes16
(around 1 s-1 100 µm-1). While Iso led to a ~4 fold increase in Ca2+ spark frequency, this
was not observed with forskolin and was not prevented by the PKA inhibitor PKI. (D)
Ca2+ current in Ctrl and after ~3 min Iso or forskolin. For this experiment the cells were
held at -40 mV to inactivate Na+ currents. Normalized Ca2+ current in Iso (n=5, N=4) or
forskolin (n=6, N=5). Current stimulation by forskolin was similar to that by Iso,
documenting comparable PKA activation. (E) Typical SR Ca2+ content assessment by 10
mM caffeine. In forskolin, SR content is increased to 125% ± 8.8% (n=15, N=5) because
of SERCA stimulation without activation of sparks. In contrast, in PKI the SR Ca2+
content decreased, partly because PKA inhibition prevents SERCA stimulation.
Figure 2 CaMKII is involved in spark frequency modulation, but not via ROS-dependent
CaMKII activation. (A) Line-scan images in Ctrl and after 1 µM Iso, both in the presence
of 10 µM of AIP in the patch pipette. (B) Ca2+ spark frequency in Ctrl and in Iso, in the
absence (n=8, N=8) or presence of the CaMKII inhibitors AIP (n=9, N=5), KN-93 (n=5,
N=2) and its inactive analogue KN-92 (n=5, N=3). (C) Unchanged SR Ca2+ content in the
presence of AIP, KN-93 and KN-92. (D) Line-scan images recorded after 1 hour pre-
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incubation with 100 µM ROS scavenger Mn-TBAP in Ctrl and after ~3 min of 1 µM Iso.
(E) Ca2+ spark frequency during Iso without (n=8, N=8) and with Mn-TBAP (n=5, N=5 ).
(F) Unchanged SR Ca2+ content in the presence of the ROS scavenger Mn-TBAP.
Figure 3 The increase of Ca2+ spark frequency is dependent on NO. (A) Comparison of
the Ca2+ spark frequency stimulation by 1 µM Iso without (n=8, N=8) and with (n=7, N=5)
1 hour pre-incubation with 500 µM L-NAME. (B) SR Ca2+ content remained unchanged in
the presence of L-NAME. (C) Representative signal of DAF-2 fluorescence before (Ctrl)
and after ~3 min of 1 µM Iso. (D) Representative time-course of NO production upon 1
µM Iso application. At the end, GSNO is applied as a positive control for NO detection.
(E) Averaged time-course of DAF-2 fluorescence in Ctrl and during Iso recorded in the
absence (red trace n=7, N=6) and presence of L-NAME (orange trace, n=6, N=4). (F)
Normalized NO-induced DAF-2 fluorescence upon Iso (red bar) and after pre-incubation
in 500 µM L-NAME (orange bar). L-NAME prevented the NO signal observed in control,
confirming that the DAF-2 fluorescence resulted from NO production.
Figure 4. The NO donor GSNO increases Ca2+ spark frequency upon CaMKII activation
via nitrosation. (A) Line-scan images of Ca2+ spark recordings in Ctrl (top), during 150
µM GSNO alone (middle) and GSNO in the presence of 10 µM AIP in the patch pipette
(bottom). (B) Normalized spark frequencies after ~3 min of 1 µM Iso (blue bar n=6, N=6 )
and GSNO alone (red bar n=14 N=11) and with 10 µM AIP (white bar n=9 N=4).
Inhibiting CaMKII with AIP prevented the higher frequency induced by GSNO. (C) After
GSNO, the SR Ca2+ content was not maintained (83% ±6% of control) due to a higher
spark frequency without SERCA stimulation in parallel. (D) Quantitative in-vitro CaMKII
activation in response to 1 µM H2O2 (blue bar) or 500 µM GSNO (red bar) in comparison
to control (white bar). CaMKII activities were normalized to full Ca2+/CaM-dependent and
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control activities (N=5). (E) Detection of nitrosated CaMKII in-vitro by anti-Cys-SNO
specific antibody reveals increased nitrosation in GSNO.
Figure 5. In beating cardiomyocytes, the NO donor GSNO increased diastolic Ca2+
spark frequency and induced arrhythmogenic diastolic Ca2+ waves. (A) Protocol used to
record Ca2+ transients and Ca2+ waves from myocytes during and immediately after
stimulation, with or without the presence of GSNO. (B) GSNO resulted in more diastolic
Ca2+ sparks,(C) in a higher propensity for spontaneous Ca2+ waves (SCWS) and (D) in
reduced SR Ca2+ content. (E) Statistical analysis of SCWS (n=9, N=5) and SR content
(n=9, N=5).
Figure 6. Diagram of the involved pathways during β-AR stimulation of resting
cardiomyocytes. Our results show that during β-AR stimulation by Iso, endogenous
production of NO derived from NOS activates CaMKII and subsequently modulates the
RyRs open probability, as reflected by the higher Ca2+ spark frequency.
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